The alteration in bone metabolism with increasing age was investigated in the femoral diaphysis of male rats. Calcium content was highest in the bone from 3-week-old rats (491±13 mg/g bone ash), falling gradually with aged to 357±7 and 306±9 mg/g bone ash in 28- and 52-week-old rats, respectively. Bone zinc content increased until rats were 3 weeks of age, and thereafter remained constant. Deoxyribonucleic acid (DNA) content was highest in the bone from 1-week-old rats, and it decreased markedly with increasing age. Alkaline phosphatase and acid phoshatase activities increased up to 3 weeks, then subsequently declined with age. Thus, the retardation of bone metabolism was induced by ageing. When zinc sulfate (5.0, 10.0 and 20.0 mg Zn/kg body weight) was administered orally for 3 d to 28-week-old rats, alkaline phosphatase activity and calcium content in the femoral diaphysis was elevated markedly by all doses. The oral administration of vitamin D₃ (2.0 and 20 μg/kg) for 3 d in 28-week-old rats did not produce an appreciable increase in bone alkaline phosphatase activity or calcium content, while 1, 25-dihydroxyvitamin D₃ (1.5 μg/kg) caused a significant increase in those biochemical indices. These results indicate that zinc and 1, 25-dihydroxyvitamin D₃ play a role as activators in bone metabolism of ageing rats.

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A previously reported fluorometric high performance liquid chromatographic procedure for separation of 9-anthryldiazomethane (ADAM) derivatives of prostaglandins (PG's) was applied to examine catabolic products of PG's, such as 15-keto-PGE₂, 13, 14-dihydro-15-keto-PGE₂, 15-keto-PGF_2α, and 13, 14-dihydro-15-keto-PGF_2α. ADAM derivatives of these metabolites were separated in 30 min on an ODS column with 65% acetonitrile. By this method catabolism of PGE₂ and PGF_2α in kidney homogenates of rats and rabbits was analyzed. Homogenates of medulla and cortex of rat kidney catabolized PGE₂ and PGF_2α to 15-keto-PG's. Whereas the homogenate of rabbit kidney cortex yielded 13, 14-dihydro-15-keto-PGE₂, that of medulla did not. The conversion of PGF_2α to PGE₂ was shown to occur in homogenates of rabbit kidney cortex as well as rat kidney medulla and cortex.

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The effect of formulations of lecithin-dispersed preparation on the absorption of d-α-tocopherol acetate (VEA) from the small intestine was investigated in rats. When lecithin-dispersed preparations containing VEA or polysorbate 80 (PS-80)-solubilized solution of VEA were intraduodenally administered, VEA was hydrolyzed to d-α-tocopherol (VE) and was not detected in the plasma nor in the thoracic lymph. The maximum plasma concentration (C_max) of VE after the intraduodenal administration of a preparation consisting of VEA, soybean phosphatidylcholine (PC) and medium-chain triglycerides (MCTG) (VEA/PC/MCTG, 5/16/1 by weight) was highest among the VEA preparations, and PS-80-solubilized solution gave the lowest C_max. AUC of VE up to 24 h was also increased by the addition of MCTG to VEA/PC preparation. In the thoracic duct-fistula rat, the transport of VE into the thoracic lymph was increased by the administration of the VEA/PC/MCTG preparation significantly more than the VEA/PC preparation ; the cumulative amounts of VE recovered in the thoracic lymph up to 24 h were 23.2±0.5% and 10.9±1.5% of dose, respectively. The plasma concentration of VE was not increased in the thoracic duct-fistula rat even after the intraduodenal administration of VEA preparations, suggesting that VE is not transported directly to the systemic circulation, but by way of the lymphatic route. The lymphatic transport of VE following the intraduodenal administration of VEA/PC/MCTG preparation was markedly diminished by the simultaneous administration of Pluronic L-81 emulsion, an inhibitor of chylomicron formation. It is suggested that the chylomicron is essential to the lymphatic transport of VE from VEA preparations.

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Forskolin synergistically potentiated adenosine 3', 5'-cyclic monophosphate formation by prostaglandin E₁ (PGE₁) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE₁ in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE₁, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimido-diphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl₂] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE₁-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

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In order to determine the specific action of cadmium on bone metabolism, the effect of cadmium on alkaline phosphatase activity, a marker enzyme of osteoblasts, was compared with that of other divalent heavy metal ions, i.e., zinc, manganese, lead, copper, nickel and mercury (10 μM each), using cloned osteoblast-like cells, MC3T3-E1. Cadmium had the strongest inhibitory effect on alkaline phosphatase activity of the cells among the metals tested. At the same dose, however, cadmium failed to inhibit cellular glucose-6-phosphatase and lactate dehydrogenase activities, suggesting that the inhibitory effect of cadmium on alkaline phosphatase was specific and was not dependent on cell injury. Cadmium treatment caused a significant decrease in cellular zinc level, but mercury treatment had no such effect at the dose inhibiting alkaline phosphatase activity. There was a good correlation between decrease of cellular zinc level and inhibition of alkaline phosphatase activity in cadmium-treated cells. Concomitant treatment of the cells with zinc prevented the cadmium-induced inhibition of alkaline phosphatase activity. However, this was not the case in the mercury-induced inhibition. Cadmium also inhibited the mineralization of osteoblasts. When 10 or 20 μM zinc was concomitantly added to the cultures, the inhibition of mineralization was prevented. These data suggest that the inhibitory effect of cadmium in osteoblasts may be closely related to its influence on the cellular zinc metabolism.

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MRL/Mp-lpr/lpr (MRL/1) mice spontaneously develop autoimmune kidney disease which resembles human systemic lupus erythematosus (SLE). Employing this strain of mouse, we examined the effect of several immunosuppressants and a newly synthetized anti-nephritic agent, 4-chloro-3', 6'-dimethyl-2, 2'-iminodibenzoic acid (TO-115) on both the development of immunological abnormalities and the clinical symptoms of autoimmune kidney disease. This study aimed to determine how much the magnitude of autoantibody suppression related to the degree of prevention of autoimmune nephritis. Immunological abnormalities were assessed by measuring the serum levels of anti-deoxyribonucleic acid and anti-trinitrophenyl antibodies, rheumatoid factor (RF), and immune complex (IC). The status of autoimmune kidney disease was monitored by means of the appearance of proteinuria and survival time and the measurement of serum levels of blood urea nitrogen (BUN) and cholesterol. Immunosuppressants such as cyclophosphamide (CY), 6-mercaptopurine (6MP) and sodium aurothiomalate (SAT) remarkably suppressed the development of immunological abnormalities in a dose dependent manner. Interestingly, however, only CY showed the suppressive effect on the development of autoimmune kidney disease with prolongation of survival time and the excretion of proteinuria. In contrast, 6-MP and SAT did not inhibit the development of autoimmune kidney disease. On the other hand, TO-115 did not suppress the development of immunological abnormalities, but it restrained the symptoms of autoimmune kidney disease. Taken together, the suppression of autoantibody production does not always lead to prevention of the development of autoimmune kidney disease. It seems likely that either complete suppression of antibody production or sufficient repression of subsequent IC-induced inflammation is essential to prevent the development of autoimmune kidney disease, and to prolong the lifespan of MRL/l mice.

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Using high performance liquid chromatography with electrochemical detection, the levels of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), 3, 4-dihydroxyphenylacetic acid (DOPAC) and 3, 4-dihydroxyphenylalanine (DOPA) were determined in various brain regions of the rat 1 h after nicotine administration. Nicotine (1 mg/kg, s.c.) produced an increase in MHPG concentrations in the occipital cortex, hippocampus, striatum, hypothalamus, thalamus, midbrain, pons/medulla and cerebellum. This alkaloid at the same dose also caused an increase in DOPAC concentrations in the hypothalamus, thalamus and pons/medulla. The nicotine-induced increase in MHPG and DOPAC concentrations in the brain regions was inhibited by pretreatment with mecamylamine (5 mg/kg, i.p.) but not by hexamethonium (10 mg/kg, i.p.). Nicotine (1 mg/kg, s.c.) produced an increase in DOPA concentrations under DOPA decarboxylase inhibition with NSD-1015 (200 mg/kg, i.p.) in the hypothalamus, thalamus and pons/medulla. These results indicate that nicotine can increase the turnover of noradrenaline and dopamine in various brain regions of the rat and this effect is mediated via activation of central nicotinic receptors.

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We have examined effects of quaternary ammonium compounds on the in vitro degradation of endogenous lipids in isolated lysosomes. The degree of lipid degradation was assessed by determining hydrolysis products of labeled lipid. Lipolysis was inhibited by quaternary ammonium compounds. The degrees of inhibition were as follows : ethidium bromide>N-methylatropinium bromide (NMA)>tubocurarine. The inhibition of lipolysis by these quaternary ammonium compounds is not necessarily correlated with the differences in their polarties, molecular weight or structures. The degradation of three phospholipid classes was inhibited by NMA with phosphatidylcholine the most vulnerable. The effect of NMA on the hydrolysis of [¹⁴C] dipalmitoylphosphatidylcholine (phospholipid) by lysosomal soluble proteins was also examined. The effect of NMA on phospholipase A₁ was assessed by the formation of lysophosphatidylcholine, and that on phospholipase C was assessed as the sum of mono-and diglyceride formations. The action of NMA on the phospholipid degradation was similar to that of cationic amphiphilic drugs, but it differed somewhat from that of chloroquine for each enzyme. From these results, it was concluded that one of the inhibitory mechanisms of phospholipid degradation by NMA was the direct interaction between NMA and phospholipase A₁ or C.

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Antitumor activity of Sarcodon aspratus (BERK.) S. ITO and Ganoderma lucidum (FR.) KARST. was investigated. Methanol and aqueous extracts of these Japanese mushrooms were tested for antitumor activity against solid type of sarcoma 180 by intraperitoneal or oral administration. The aqueous extract was remarkably effective for inhibition of tumor growth, but the methanol extract was not. The fraction of molecular weight more than 10000 had a high inhibitory activity against the tumor growth, but the fraction of molecular weight less than 10000 did not. Fractionation was carried out by using an ion-exchanger, and fraction S-4 having the highest carbohydrate content had the highest antitumor activity by intraperitoneal administration.

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